Technical field
[0001] The present disclosure relates to a use of a methylation site as a disease diagnosis
marker, in particular relates to a use of a methylation site in a Y chromosome as
a prostate cancer diagnosis marker.
Background
[0002] DNA methylation is a common epigenetic modification. Under the catalysis of DNA methyltransferase,
the carbon atom at the 5th position of cytosine is methylated using the methyl group
provided by S-adenosylmethionine, thereby transforming cytosine to 5-methylcytosine,
which plays a vital role in the regulation of gene expression.
[0003] Cancer, also known as malignant tumor, is a disease caused by the abnormalities in
the control of cell growth and proliferation mechanisms. Under normal circumstances,
the growth and proliferation of cells are strictly regulated by some genes regulating
the growth and development, including the oncogenes and the cancer suppressor genes,
to maintain a normal physiological state; when affected by multiple factors such as
radiation, chemical contamination, viral infection and endocrine imbalance, the activation
of oncogenes and inactivation of tumor suppressor genes in vivo will be caused, thereby
leading to the occurrence of cancer. In addition to the gene mutations, abnormal regulations
of the epigenetics such as histone modification, DNA methylation also play an important
role during the occurrence and development of tumor, the abnormalities of epigenetic
modification can be found in a variety of cancers. In short, the occurrence of tumor
is an extremely complex process, it is the result of abnormal regulation and its accumulation
occurred in oncogenes, cancer suppressor genes and epigenetic modification in vivo.
[0004] DNA methylation has a close relation with the occurrence of cancer, the abnormalities
of DNA methylation have been found in many cancers. DNA methylation has a certain
degree of stability, it is an early event during the occurrence of cancer. In recent
years, many studies have shown that abnormalities of DNA methylation can be used as
a biomarker for cancer diagnosis. At present, abnormalities of DNA methylation in
many specific gene promoter regions are used as a potential marker for the diagnosis
for prostate cancer.
[0005] At present, the main diagnostic methods for prostate cancer are rectal diagnosis,
serum PSA examination, and tissue immune biopsy, etc., in which the tissue immune
biopsy is the gold diagnostic method for prostate cancer. However, since it is required
to take the prostate tissue from a patient firstly, which will bring about great pains
for the patient, it is not suitable for the previous diagnosis. PSA is currently used
as a marker for the previous diagnosis for prostate cancer, but it is not quite accurate
in terms of examination, and a certain gray zone exists. Therefore, it is very urgent
to find a convenient and accurate diagnosis marker for the early diagnosis for prostate
cancer.
[0006] Meanwhile, there are various methods for monitoring the DNA methylation in the prior
art, for example, a quantitative analysis method for monitoring the DNA methylation
disclosed in
CN 104062334A, as another example, a DNA methylation chip technology. However, at the present stage,
there exists a lack of a unified and more accurate criterion when determining whether
the methylation is abnormal, therefore it is unable to predict the risk for cancer
as early as possible through the change of the methylation level at specific sites.
Summary of the Disclosure
[0007] The inventors of the present disclosure have established a unified, standard method
for screening a disease-related methylation site and screened out a methylation site
in a Y chromosome associated with prostate cancer, whereby completing the present
disclosure.
[0008] The first aspect of the present disclosure relates to a use of a reagent for detecting
a methylation level of a methylation site in a Y chromosome of a sample to be tested
of a subject in the preparation of a kit, the kit is used for one or more application(s)
selected from the group consisting of: diagnosis for prostate cancer, assessment of
a risk for prostate cancer, evaluation of a therapeutic effect on prostate cancer,
and screening of a therapeutic drug for prostate cancer; the methylation site in a
Y chromosome is one or more selected from the group consisting of cg03052502, cg04462340,
cg05163709, cg05544622, cg14466580 and cg27539833.
[0009] In some embodiments, the sample to be tested is selected from the group consisting
of tissue, urine (e.g., urine after prostate massage) and prostatic fluid.
[0010] In some embodiments, the sample to be tested is selected from the group consisting
of urine (e.g., urine after prostate massage) and prostatic fluid.
[0011] In some embodiments, the methylation site in a Y chromosome is selected from the
group consisting of cg05163709 and cg27539833.
[0012] In some embodiments, the method for detecting a methylation level of a methylation
site in a Y chromosome of a sample to be tested of a subject is selected from the
group consisting of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative
methylation-specific polymerase chain reaction, southern blotting, restriction landmark
genomic scanning, single nucleotide primer extension, CpG island microarray, single
nucleotide primer extension (SNUPE), combined sodium bisulfite restriction endonuclease
analysis and mass spectrometry.
[0013] In some embodiments, the reagent is an oligonucleotide primer which is used for amplifying
a nucleotide sequence comprising a methylation site in a Y chromosome.
[0014] The second aspect of the present disclosure relates to a kit comprising a reagent
for detecting a methylation level of a methylation site in a Y chromosome of a sample
to be tested of a subject, the kit is used for one or more application(s) selected
from the group consisting of: diagnosis for prostate cancer, assessment of a risk
for prostate cancer, evaluation of a therapeutic effect on prostate cancer, and screening
of a therapeutic drug for prostate cancer; the methylation site in a Y chromosome
is one or more selected from the group consisting of cg03052502, cg04462340, cg05163709,
cg05544622, cg14466580 and cg27539833.
[0015] In some embodiments, the sample to be tested is selected from the group consisting
of tissue, urine (e.g., urine after prostate massage) and prostatic fluid.
[0016] In some embodiments, the sample to be tested is selected from the group consisting
of urine (e.g., urine after prostate massage) and prostatic fluid.
[0017] In some embodiments, the methylation site in a Y chromosome is selected from the
group consisting of cg05163709 and cg27539833.
[0018] In some embodiments, the method for detecting a methylation level of a methylation
site in a Y chromosome of a sample to be tested of a subject is selected from the
group consisting of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative
methylation-specific polymerase chain reaction, southern blotting, restriction landmark
genomic scanning, single nucleotide primer extension, CpG island microarray, single
nucleotide primer extension (SNUPE), combined sodium bisulfite restriction endonuclease
analysis and mass spectrometry.
[0019] In some embodiments, the reagent is an oligonucleotide primer which is used for amplifying
a nucleotide sequence comprising a methylation site in a Y chromosome.
[0020] The third aspect of the present disclosure relates to a method for diagnosis for
prostate cancer, assessment of a risk for prostate cancer, evaluation of a therapeutic
effect on prostate cancer, and screening of a drug for prostate cancer, the method
comprises a step of detecting a methylation level of a methylation site in a Y chromosome
of a sample to be tested of a subject; the methylation site in a Y chromosome is one
or more selected from the group consisting of cg03052502, cg04462340, cg05163709,
cg05544622, cg14466580 and cg27539833.
[0021] In some embodiments, when the methylation level of the methylation site selected
from one or more of cg03052502, cg04462340, cg05544622, cg14466580 and cg27539833
is decreased as compared to a normal sample or a normal reference value, it is indicated
that the subject has suffered from prostate cancer or a high risk for prostate cancer;
when the methylation level of the methylation site of cg05163709 is increased as compared
to a normal sample or a normal reference value, it is indicated that the subject has
suffered from prostate cancer or a high risk for prostate cancer.
[0022] In some embodiments, when the methylation level of the methylation site selected
from one or more of cg03052502, cg04462340, cg05544622, cg14466580 and cg27539833
is increased as compared to that prior to treatment or use of a drug for screening,
it is indicated that the treatment for the subject is effective or the drug for screening
is effective; when the methylation level of the methylation site of cg05163709 is
decreased as compared to that prior to treatment or use of a drug for screening, it
is indicated that the treatment for the subject is effective or the drug for screening
is effective.
[0023] In some embodiments, the sample to be tested is selected from the group consisting
of tissue, urine (e.g., urine after prostate massage) and prostatic fluid.
[0024] In some embodiments, the sample to be tested is selected from the group consisting
of urine (e.g., urine after prostate massage) and prostatic fluid.
[0025] In some embodiments, the methylation site in a Y chromosome is selected from the
group consisting of cg05163709 and cg27539833.
[0026] In some embodiments, the method for detecting a methylation level of a methylation
site in a Y chromosome of a sample to be tested of a subject is selected from the
group consisting of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative
methylation-specific polymerase chain reaction, southern blotting, restriction landmark
genomic scanning, single nucleotide primer extension, CpG island microarray, single
nucleotide primer extension (SNUPE), combined sodium bisulfite restriction endonuclease
analysis and mass spectrometry.
[0027] In some embodiments, the method for detecting a methylation level of a methylation
site in a Y chromosome of a sample to be tested of a subject comprises a step of using
an oligonucleotide primer which is used for amplifying a nucleotide sequence comprising
a methylation site in a Y chromosome.
[0028] The fourth aspect of the present disclosure relates to the use of a methylation site
in a Y chromosome as a biomarker for diagnosis for prostate cancer, assessment of
a risk for prostate cancer, evaluation of a therapeutic effect on prostate cancer,
an screening of a drug for prostate cancer, the methylation site in a Y chromosome
is one or more selected from the group consisting of cg03052502, cg04462340, cg05163709,
cg05544622, cg14466580 and cg27539833.
[0029] In some embodiments, the diagnosis for prostate cancer, the assessment of the risk
for prostate cancer, the evaluation of the therapeutic effect on prostate cancer,
and the screening of the drug for treating prostate cancer can be performed by detecting
the methylation level of the methylation site in a Y chromosome.
[0030] The fifth aspect of the present disclosure relates to a biomarker for diagnosis for
prostate cancer, assessment of a risk for prostate cancer, evaluation of a therapeutic
effect on prostate cancer, and screening of a drug for prostate cancer, the biomarker
is a methylation site in a Y chromosome which is one or more selected from the group
consisting of cg03052502, cg04462340, cg05163709, cg05544622, cg14466580 and cg27539833.
[0031] In some embodiments, the diagnosis for prostate cancer, the assessment of the risk
for prostate cancer, the evaluation of the therapeutic effect on prostate cancer,
and the screening of the drug for treating prostate cancer can be performed by detecting
the methylation level of the methylation site in a Y chromosome.
[0032] The sixth aspect of the present disclosure relates to a method for screening the
methylation site in a Y chromosome associated with a disease, the method comprises:
1) obtaining the patient's disease sample and normal sample; 2) determining the information
of methylation of the chromosome in the disease sample and the normal sample; 3) screening
the methylation conserved site according to the information of methylation of the
chromosome in the normal sample; 4) screening the methylation site in the disease
sample having obvious differences from that in the normal sample according to the
information of methylation of the chromosome in the disease sample and the normal
sample; 5) combining the results from steps 3) and 4) to obtain the methylation conserved
site having obvious differences which is the methylation site in the chromosome associated
with the disease.
[0033] In some embodiments, the disease is a cancer, for example prostate cancer.
[0034] In some embodiments, the chromosome is a euchromosome or a sex chromosome (e.g.,
a Y chromosome).
[0035] In some embodiments, the sample is derived from tissue (e.g., cancerous tissue),
blood, urine, feces or tissue fluid (e.g., prostatic fluid).
[0036] In some embodiments, the methylation conserved site refers to a methylation site
having a methylation level with a standard deviation SD value of less than or equal
to 0.25 in the normal sample.
[0037] In some embodiments, the methylation site having obvious differences is a site at
which the change in the methylation level is 0.2 or more and the p value and the q
value are both less than or equal to 0.01 when the disease sample is compared with
the normal sample; the change is an increase or a decrease.
[0038] The present disclosure also relates to a method for diagnosis for prostate cancer,
the method comprises a step of screening a methylation site in a Y chromosome associated
with prostate cancer.
[0039] In some embodiments, the steps for screening the methylation site in a Y chromosome
associated with prostate cancer comprise: 1) obtaining the disease sample and normal
sample from the patient with prostate cancer; 2) determining the information of methylation
of the Y chromosome in the disease sample and the normal sample; 3) screening the
methylation conserved sites according to the information of methylation of the Y chromosome
in the normal sample; 4) screening the methylation site in the disease sample having
obvious differences from that in the normal sample according to the information of
methylation of the Y chromosome in the disease sample and the normal sample; 5) combining
the results from steps 3) and 4) to obtain the methylation conserved site having obvious
differences which is the methylation site in the chromosome associated with prostate
cancer.
[0040] In some embodiments, the sample is derived from tissue (e.g., cancerous tissue),
blood, urine (e.g., urine after prostate massage), feces or tissue fluid (e.g., prostatic
fluid).
[0041] In some embodiments, the methylation conserved site refers to a methylation site
having a methylation level with a standard deviation SD value of less than or equal
to 0.25 in the normal sample.
[0042] In some embodiments, the methylation site having obvious differences is a site at
which the change in the methylation level is 0.2 or more and the p value and the q
value are both less than or equal to 0.01 when the disease sample is compared with
the normal sample; the change is an increase or a decrease.
[0043] In the present disclosure, the "diagnosis", "assessment of a risk for a disease"
and "evaluation of a therapeutic effect" have the meaning well-known in the art. For
example, the "diagnosis" refers to a judgment on whether one suffers from the disease;
the "assessment of a risk for a disease" refers to an assessment on the magnitude
of a risk for suffering from a disease and the risk for recurrence after treatment;
the "evaluation of a therapeutic effect" refers to an evaluation on whether there
is any therapeutic effect, for example, if the symptoms are relieved, disappear, the
size of focus is reduced or disappear, or the disease is no longer progressing, the
treatment is effective.
[0044] In the present disclosure, the "disease sample" refers to a tissue sample in which
the focus is located or a tissue sample associated with the disease, for example,
a disease sample of prostate cancer includes both the cancerous tissue samples of
prostate cancer and the prostatic fluid samples and urine samples associated with
prostate cancer, especially urine samples after prostate massage.
[0045] In the present disclosure, the "normal sample" and the "normal specimen" have the
same meaning and refer to a sample of a normal tissue opposite to the "disease sample".
For example, when the disease sample is a cancerous tissue sample, the normal sample
may be a paracancerous tissue sample; and when the disease sample is a diseased blood
or urine sample, the normal sample may be a pre-diseased blood or urine sample.
[0046] In the present disclosure, the "normal reference value" has a meaning well-known
in the art and refers to a normal value range of an index obtained on the basis of
a certain number of normal samples, for example, on the basis of a certain number
of normal samples, the normal reference value range for a methylation level of a methylation
site in a Y chromosome can be obtained. When determining whether an index of a sample
is within the normal range, this normal reference value has reference significance.
[0047] In the present disclosure, the kit may further contain one or more reagent(s) selected
from the group consisting of dNTP, buffer, DNA polymerase, restriction endonuclease
(including methyl-specific endonuclease), and marker (including fluorescent marker,
chemiluminescent marker and radioactive marker, etc.).
[0048] In the present disclosure, the method for designing an oligonucleotide primer for
detecting a methylation level of a methylation site based on the methylation site
on the genome is well known in the art, and the oligonucleotide primer can be complementary
to or hybridized with the nucleic acid sequence at which the methylation site is located.
[0049] In the present disclosure, the number of the oligonucleotide primer is at least one
or a group of primers, for example, at least 2, at least 3, at least 4, at least 5,
at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least
12, which are complementary to or hybridized with the nucleic acid sequence at which
the methylation site is located.
[0050] In a specific embodiment of the present disclosure, the primers for amplifying the
site cg05163709 are:
F: GGAAAGGGGTGATTAAATATTTAGTTA (SEQ ID NO: 1);
R: 5'-BIOTIN-CAACCTAATAAAAAACTATACAAACACAT (SEQ ID NO: 2);
S-primer: ATAAGTATGTTTAATTATTGTTTAAG (SEQ ID NO: 3).
[0051] In a specific embodiment of the present disclosure, the primers for amplifying the
site cg27539833 are:
F: GGAATAGTTTAGTTAAAGAAAAAGGTTAAGAT (SEQ ID NO: 4);
R: 5'-BIOTIN-AATTTACCACAATACACAAAAAACTAACTACTTA (SEQ ID NO: 5);
S-primer: AGATTTTAGTAGTTTTTTGTCGTTA (SEQ ID NO: 6).
[0052] In the present disclosure, the representing method "cg" for a methylation site in
a chromosome is based on the nomenclature for each methylation site by 450K chip from
Illumina Corporation. The meaning of the representing method is well known in the
art and the methylation site represented thereby one to one corresponds to a specific
methylation site in the chromosome, and one skilled in the art can accurately and
uniquely match a site represented according to this method onto a human chromosome.
[0053] In the present disclosure, the "methylation" refers to the methylation of the CpG
site.
[0054] In the present disclosure, the methylation level may be represented by a method well
known in the art, for example it can be represented by the ratio of methylated cytosine
C detected to the total C detected at the time of sequencing, specifically β value
(beta value), and the numerical range thereof is 0-1 or 0-100%. In some embodiments,
the representing method for methylation level is β value (beta value), and the numerical
range thereof is 0-1.
[0055] In the present disclosure, the method for determining the increase or decrease of
the methylation level is the difference value of β values (for example, the difference
value between samples) is greater than or equal to 0.2.
[0057] In the present disclosure, "p value" and "q value" have the meanings and calculation
methods well known in the art, for example one can refer to "Biometry and Experimental
Design" , for example p value = probability of ones assumed to be correct but rejected
= number of negative ones/total number, which is an inspection probability for a sample
data; q value = probability of ones rejected but correct = number of false positive
ones/number of ones presumably positive, which is an inspection probability of the
deduction you obtained, and is calculated based on the P value. It can be said that
the q value is a further statistic of the p value.
Beneficial effects of the disclosure
[0058] The present disclosure has established a method and criterion for screening a methylation
site in a chromosome associated with a disease, it also screens out six methylation
sites in the Y chromosome associated with the prostate cancer diagnosis by using prostate
cancer as an example. The screened methylation sites in the Y chromosome can be used
for early and rapid diagnosis for the disease as a prostate cancer diagnosis marker.
Brief Description of the Drawings
[0059]
Fig. 1 shows the thermal map of the methylation level of 75 conserved sites screened
out from the paracancerous tissue.
Fig. 2 shows the 37 methylation sites having significant changes screened out through
the comparison of the cancer tissue with the paracancerous tissue.
Fig. 3 and Fig. 4 show the 75 methylation sites conserved in the paracancerous tissue
(Fig. 3), wherein 6 methylation sites have significant changes in the cancerous tissue
(Fig. 4).
Fig. 5 shows the methylation level of the final 6 sites screened out (the Figure above),
and the proportion of the occurrence of significant change of each site in 66 cancerous
tissues (the Figure below, the black mark represents the significant change).
Fig. 6 shows the DNA methylation level of sites cg05163709 and cg27539833 in the positive
and negative prostate puncture biopsies through the detection for a urine sample.
Fig.7 shows the comparison of diagnostic efficiency between the methylation of sites
cg05163709 and cg27539833 as a diagnosis marker and the PSA.
Detailed Description
[0060] Although the embodiments of the present disclosure will be described in detail with
reference to the following examples, it will be understood by those skilled in the
art that the following examples are merely intended to be illustrative of the present
disclosure and are not to be construed as limiting the scope of the present disclosure.
The Examples in which the specific conditions are not specified are carried out according
to the conventional conditions or conditions suggested by the manufacturer. The used
reagents or instruments of which the manufacturers are not indicated, are all commercially
available conventional products.
Example 1 Collection and identification of cancerous tissues and paracancerous tissues
of prostate cancer
[0061] The tissues were collected from prostate cancer patients undergoing cancer cutting
operation, the cancerous tissues and paracancerous tissues were distinguished through
tissue immune biopsy by an experienced physician, and collected correspondingly (the
samples were provided by Shanghai Changhai Hospital).
Example 2 Screening of conserved methylation sites
[0062] Paracancerous tissues from 66 cases of patients with prostate cancer were collected
and taken, and DNAs were extracted therefrom and amplified (QIAamp DNA Mini Kit (Cat.No.51306)).
Whole genome methylation levels of 66 samples were measured using the DNA methylation
chip Illumina 450K (Infinium HumanMethylation450 BeadChip array), the initial data
obtained by scanning were processed through the GenomeStudio software according to
Illumina's official Methylation Analysis Algorithms to generate the data containing
the methylation level at each site for each sample, i.e., Raw data, then the methylation
levels of sites after filtration, i.e., Norm data, were given upon the correction
and normalization of deviation caused by different types of fluorescence and probe,
and site filtration, and the methylation level of each site was represented with β
value (0-1). The methylation information in a Y chromosome therein was taken, subjected
to analysis and comparision, and 75 methylation conserved sites (SD value ≤ 0.25)
were screened out according to the SD values of the methylation levels (β value) between
samples. The results were shown in Fig. 1.
Example 3 Screening of methylation sites having obvious differences
[0063] 37 DNA methylation sites having significant changes in the cancerous tissues were
screened out, i.e., the site with a Δβ ≥ 0.2 (i.e., the difference value of β value
between the cancerous tissues and paracancerous tissues ≥0.2) and a p value ≤0.01
according to the comparison of DNA methylation levels (β value) in a Y chromosome
between 66 pairs of cancerous tissues and paracancerous tissues using the Illumina
Methylation Analyzer (IMA) software package, see Fig. 2.
Example 4 Screening of conserved sites having significant changes
[0064] According to the results in Example 2 and Example 3, the intersections of both (i.e.,
the methylation sites which were conserved in the paracancerous tissues and had significant
changes in the cancerous tissues) were screened out, there were a total of 6 sites
of cg03052502, cg04462340, cg05163709, cg05544622, cg14466580 and cg27539833 (see
Fig. 3 and Fig.4), and the specific information of these 6 sites was as shown in Table
1.
![](https://data.epo.org/publication-server/image?imagePath=2017/48/DOC/EPNWA1/EP15878635NWA1/imgb0002)
[0065] These 6 sites were compared and detected in each pair of cancerous tissue-paracancerous
tissue to calculate the proportion of the cancerous tissue which indeed had a significant
change in the methylation (see Fig. 5). These 6 intersection sites were the conserved
sites having significant changes in the methylation level in the cancerous tissues
and can be used as markers for the diagnosis for prostate cancer.
Example 5 Detection of methylation levels of sites cg05163709 and cg27539833 in the
urine sample
[0066] The urine samples were obtained from patients with prostate cancer and normal subjects
through the prostate massage, and DNAs were extracted therefrom. The methylation levels
of sites cg05163709 and cg27539833 were obtained with pyrosequencing, the changes
in methylation levels of these two sites in the urine samples were compared and analyzed
between the negative and positive specimens from prostate puncture biopsies, as shown
in Fig. 7.
[0067] The sequence of each primer is as follows:
cg05163709: F: GGAAAGGGGTGATTAAATATTTAGTTA (SEQ ID NO: 1);
R: 5'-BIOTIN-CAACCTAATAAAAAACTATACAAACACAT (SEQ ID NO: 2);
S-primer: ATAAGTATGTTTAATTATTGTTTAAG (SEQ ID NO: 3). Cg27539833
F: GGAATAGTTTAGTTAAAGAAAAAGGTTAAGAT (SEQ ID NO: 4);
R: 5'-BIOTIN-AATTTACCACAATACACAAAAAACTAACTACTTA (SEQ ID NO: 5);
S-primer: AGATTTTAGTAGTTTTTTGTCGTTA (SEQ ID NO: 6).
Example 6 The superior diagnostic efficiency of the methylation of site cg05163709
as a diagnosis marker over PSA
[0068] According to the experimental results of Example 5, the diagnostic efficiencies of
the methylation of sites cg05163709 and cg27539833 as diagnosis markers were analyzed
by the receiver operating curve (ROC), and it was found that although the area under
the curve ROC (AUC) of site cg27539833 (0.729) had no obvious advantage as compared
with that of PSA (0.753), the AUC of site cg05163709 (0.944) was significantly superior
to that of PSA (0.753), therefore it had higher sensitivity (93.9%) and specificity
(83.3%) as a diagnosis marker of prostate cancer.
[0069] While the specific embodiments of the present disclosure have been described in detail,
those skilled in the art will appreciate that according to all teachings that have
been disclosed, various modifications and substitutions can be made to those details,
all of which are within the protection scope of the present disclosure. The full scope
of the present disclosure is given by the appended claims and any equivalents thereof.
![](https://data.epo.org/publication-server/image?imagePath=2017/48/DOC/EPNWA1/EP15878635NWA1/imgb0004)
1. A use of a reagent for detecting a methylation level of a methylation site in a Y
chromosome of a sample to be tested of a subject in the preparation of a kit, the
kit is used for one or more application(s) selected from the group consisting of:
diagnosis for prostate cancer, assessment of a risk for prostate cancer, evaluation
of a therapeutic effect on prostate cancer, and screening of a therapeutic drug for
prostate cancer; the methylation site in a Y chromosome is one or more selected from
the group consisting of cg03052502, cg04462340, cg05163709, cg05544622, cg14466580
and cg27539833.
2. The use of claim 1, wherein the sample to be tested is selected from the group consisting
of tissue, urine (e.g., urine after prostate massage) and prostatic fluid.
3. The use of claim 2, wherein the sample to be tested is selected from the group consisting
of urine (e.g., urine after prostate massage) and prostatic fluid.
4. The use of claim 3, wherein the methylation site in a Y chromosome is selected from
the group consisting of cg05163709 and cg27539833.
5. The use of claim 1, wherein the method for detecting a methylation level of a methylation
site in a Y chromosome of a sample to be tested of a subject is selected from the
group consisting of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative
methylation-specific polymerase chain reaction, southern blotting, restriction landmark
genomic scanning, single nucleotide primer extension, CpG island microarray, single
nucleotide primer extension (SNUPE), combined sodium bisulfite restriction endonuclease
analysis and mass spectrometry.
6. The use of claim 1, wherein the reagent is an oligonucleotide primer which is used
for amplifying a nucleotide sequence comprising methylation site in a Y chromosome.
7. A kit comprising a reagent for detecting a methylation level of a methylation site
in a Y chromosome of a sample to be tested of a subject, the kit is used for one or
more application(s) selected from the group consisting of: diagnosis for prostate
cancer, assessment of a risk for prostate cancer, evaluation of a therapeutic effect
on prostate cancer, and screening of a therapeutic drug for prostate cancer; the methylation
site in a Y chromosome is one or more selected from the group consisting of cg03052502,
cg04462340, cg05163709, cg05544622, cg14466580 and cg27539833.
8. The kit of claim 7, wherein the sample to be tested is selected from the group consisting
of tissue, urine (e.g., urine after prostate massage) and prostatic fluid.
9. The kit of claim 8, wherein the sample to be tested is selected from the group consisting
of urine (e.g., urine after prostate massage) and prostatic fluid.
10. The use of claim 9, wherein the methylation site in a Y chromosome is selected from
the group consisting of cg05163709 and cg27539833.
11. The kit of claim 7, wherein the method for detecting a methylation level of a methylation
site in a Y chromosome of a sample to be tested of a subject is selected from the
group consisting of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative
methylation-specific polymerase chain reaction, southern blotting, restriction landmark
genomic scanning, single nucleotide primer extension, CpG island microarray, single
nucleotide primer extension (SNUPE), combined sodium bisulfite restriction endonuclease
analysis and mass spectrometry.
12. The kit of claim 7, wherein the reagent is an oligonucleotide primer which is used
for amplifying a nucleotide sequence comprising a methylation site in a Y chromosome.
13. A method for diagnosis for prostate cancer, assessment of a risk for prostate cancer,
evaluation of a therapeutic effect on prostate cancer, and screening of a drug for
prostate cancer, the method comprises a step of detecting a methylation level of a
methylation site in a Y chromosome of a sample to be tested of a subject; the methylation
site in a Y chromosome is one or more selected from the group consisting of cg03052502,
cg04462340, cg05163709, cg05544622, cg14466580 and cg27539833.
14. The method of claim 13, wherein when the methylation level of the methylation site
selected from one or more of cg03052502, cg04462340, cg05544622, cg14466580 and cg27539833
is decreased as compared to a normal sample or a normal reference value, it is indicated
that the subject has suffered from prostate cancer or a high risk for prostate cancer;
when the methylation level of the methylation site of cg05163709 is increased as compared
to a normal sample or a normal reference value, it is indicated that the subject has
suffered from prostate cancer or a high risk for prostate cancer.
15. The method of claim 13, wherein when the methylation level of the methylation site
selected from one or more of cg03052502, cg04462340, cg05544622, cg14466580 and cg27539833
is increased as compared to that prior to treatment or use of a drug for screening,
it is indicated that the treatment for the subject is effective or the drug for screening
is effective; when the methylation level of the methylation site of cg05163709 is
decreased as compared to that prior to treatment or use of a drug for screening, it
is indicated that the treatment for the subject is effective or the drug for screening
is effective.
16. The method of claim 13, wherein the sample to be tested is selected from the group
consisting of tissue, urine (e.g., urine after prostate massage) and prostatic fluid.
17. The method of claim 13, wherein the sample to be tested is selected from the group
consisting of urine (e.g., urine after prostate massage) and prostatic fluid.
18. The method of claim 13, wherein the methylation site in a Y chromosome is selected
from the group consisting of cg05163709 and cg27539833.
19. The method of claim 13, wherein the method for detecting a methylation level of a
methylation site in a Y chromosome of a sample to be tested of a subject is selected
from the group consisting of pyrosequencing, bisulfite sequencing, quantitative and/or
qualitative methylation-specific polymerase chain reaction, southern blotting, restriction
landmark genomic scanning, single nucleotide primer extension, CpG island microarray,
single nucleotide primer extension (SNUPE), combined sodium bisulfite restriction
endonuclease analysis and mass spectrometry.
20. The method of claim 13, wherein the method for detecting a methylation level of a
methylation site in a Y chromosome of a sample to be tested of a subject comprises
a step of using an oligonucleotide primer which is used for amplifying a nucleotide
sequence comprising a methylation site in a Y chromosome.
21. A use of methylation site in a Y chromosome as a biomarker in diagnosis for prostate
cancer, assessment of a risk for prostate cancer, evaluation of a therapeutic effect
on prostate cancer, and screening of a drug for prostate cancer, the methylation site
in a Y chromosome is one or more selected from the group consisting of cg03052502,
cg04462340, cg05163709, cg05544622, cg14466580 and cg27539833.
22. A biomarker for diagnosis for prostate cancer, assessment of a risk for prostate cancer,
evaluation of a therapeutic effect on prostate cancer, and screening of a drug for
prostate cancer, the biomarker is a methylation site in a Y chromosome which is one
or more selected from the group consisting of cg03052502, cg04462340, cg05163709,
cg05544622, cg14466580 and cg27539833.
23. A method for screening a methylation site in a chromosome associated with a disease,
the method comprises: 1) obtaining the patient's disease sample and normal sample;
2) determining the information of methylation of the chromosome in the disease sample
and the normal sample; 3) screening a methylation conserved site according to the
information of methylation of the chromosome in the normal sample; 4) screening the
methylation site in the disease sample having obvious differences from that in the
normal sample according to the information of methylation of the chromosome in the
disease sample and the normal sample; 5) combining the results from steps 3) and 4)
to obtain the methylation conserved site having obvious differences which is the methylation
site in the chromosome associated with the disease.
24. The method of claim 23, wherein the disease is a cancer, for example, prostate cancer.
25. The method of claim 23, wherein the chromosome is a euchromosome or a sex chromosome
(e.g., a Y chromosome).
26. The method of claim 23, wherein the sample is derived from tissue (e.g., cancerous
tissue), blood, urine, feces or tissue fluid (e.g., prostatic fluid).
27. The method of claim 23, wherein the methylation conserved site refers to a methylation
site having a methylation level with a standard deviation SD value of less than or
equal to 0.25 in the normal sample.
28. The method of claim 23, wherein the methylation site having obvious differences is
a site at which the change in the methylation level is 0.2 or more and the p value
and the q value are both less than or equal to 0.01 when the disease sample is compared
with the normal sample; the change is an increase or a decrease.
29. A method for diagnosis for prostate cancer, the method comprises a step of screening
the methylation site in a Y chromosome associated with prostate cancer.
30. The method of claim 29, the step of screening the methylation site in a Y chromosome
associated with prostate cancer comprises the method of any one of claims 23-29.